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Abstract:

Stimuli-responsive magnetic nanoparticles, methods of making the
nanoparticles, and methods of using the nanoparticles.

Claims:

1. A stimuli-responsive magnetic nanoparticle having responsivity to a
magnetic fields, comprising:(a) a core having responsivity to a magnetic
field; and(b) a plurality of stimuli-responsive polymers attached to the
core, wherein the polymer terminates with a functional group capable of
covalent coupling with a capture molecule.

2. The nanoparticle of claim 1, wherein the material having responsivity
to a magnetic field comprises a metal oxide selected from the group
consisting of ferrous oxide, ferric oxide, gadolinium oxide, and mixtures
thereof.

3. The nanoparticle of claim 1, wherein the stimuli-responsive polymer
responds to a stimulus selected from the group consisting of temperature,
pH, light, electric field, and ionic strength.

4. The nanoparticle of claim 1, wherein the stimuli-responsive polymer
comprises a polymer having a balance of hydrophilic and hydrophobic
groups.

5. The nanoparticle of claim 1, wherein the stimuli-responsive polymer is
a temperature-responsive polymer.

6. The nanoparticle of claim 1, wherein the stimuli-responsive polymer
comprises polymers and copolymers of N-isopropylacrylamide.

10. The nanoparticle of claim 1, wherein the stimuli-responsive polymer is
a multi-responsive copolymer.

11. The nanoparticle of claim 1, wherein the functional group is selected
from the group consisting of carboxyl, hydroxyl, amine, ester, and
halide.

12. The nanoparticle of claim 1, wherein the core has a diameter of from
about 2 nm to about 20 nm.

13. A stimuli-responsive magnetic nanoparticle, comprising:(a) a core
having responsivity to a magnetic field; and(b) a plurality of
stimuli-responsive polymers attached to the core, wherein the polymer
terminates with a capture moiety.

14. The nanoparticle of claim 13, wherein the nanoparticle has a diameter
from about 5 nm to about 30 nm.

15. The nanoparticle of claim 13, wherein the capture moiety is selected
from the group consisting of an antibody, antigen, nucleic acid oligomer,
protein, enzyme, or enzyme substrate.

16. The nanoparticle of claim 13, wherein the capture moiety is a biotin
moiety.

17. The nanoparticle of claim 13, wherein the nanoparticle has from about
50 to about 100 biotin moieties/nanoparticle.

18. The nanoparticle of claim 16 further comprising streptavidin.

19. The nanoparticle of claim 18, wherein the nanoparticle has from about
30 to about 70 streptavidins/nanoparticle.

20. A method for capturing a diagnostic target, comprising:(a) contacting
a medium comprising a diagnostic target with a plurality of
stimuli-responsive magnetic nanoparticles, wherein each nanoparticle
comprises a capture moiety reactive toward the diagnostic target;(b)
applying an external stimulus to provide aggregated nanoparticles;(c)
subjecting the aggregated nanoparticle to a magnetic field to provide
magnetically aggregated nanoparticles; and(d) removing the stimulus and
the magnetic field to regenerate the nanoparticles, wherein the
regenerated nanoparticles further comprise the diagnostic target.

21. The method of claim 20, wherein the stimuli-responsive magnetic
nanoparticle is selected from the group consisting of a
temperature-responsive magnetic nanoparticle, a pH-responsive magnetic
nanoparticle, a light-responsive magnetic nanoparticle, an ion-responsive
magnetic nanoparticle, and a multi-responsive magnetic nanoparticle.

22. The method of claim 20, wherein the stimulus is selected from the
group consisting of temperature, pH, light, ionic strength, and
combinations thereof.

23. The method of claim 20, wherein the diagnostic target is selected from
the group consisting of an antibody, an antigen, a nucleic acid oligomer,
a protein, a polypeptide, an enzyme, or an enzyme substrate.

24. The method of claim 20, wherein the capture moiety is selected from
the group consisting of an antibody, an antigen, a nucleic acid oligomer,
a protein, a polypeptide, an enzyme, or an enzyme substrate.

25. A device, comprising(a) a channel adapted for receiving a flow
comprising a plurality of stimulus-responsive magnetic nanoparticles,
wherein the nanoparticles are reversibly self-associative in response to
a stimulus; and(b) a separation region through which the flow passes,
wherein the separation region is adapted to reversibly apply a stimulus
and a magnetic field to the flow to capture the nanoparticles.

26. An assay for detecting a diagnostic target, comprising,(a) contacting
the diagnostic target with a plurality of stimuli-responsive magnetic
nanoparticles, wherein each nanoparticle comprises a capture moiety
having affinity toward the diagnostic target;(b) forming nanoparticle
conjugates by combining the diagnostic target with the stimuli-responsive
magnetic nanoparticles;(c) aggregating the nanoparticle conjugates by
applying an external stimulus;(d) further aggregating the nanoparticle
conjugates by subjecting the aggregated nanoparticle conjugates to a
magnetic field;(e) regenerating the nanoparticle conjugates by removing
the stimulus and the magnetic field; and(f) analyzing the regenerated
nanoparticles comprising the diagnostic target.

Description:

FIELD OF THE INVENTION

[0002]The present invention relates to stimuli-responsive magnetic
nanoparticles, methods for making the nanoparticles, and methods for
using the nanoparticles.

BACKGROUND OF THE INVENTION

[0003]There has been considerable recent interest in the development of
magnetic nanoparticle (mNP) technologies for diagnostic and imaging
applications. Compared to larger magnetic particles, the smaller
nanoparticles (NPs) display potential advantages in their diffusive and
superparamagnetic properties. Magnetophoretic mobility, μm, is
defined as the acceleration of an object in the presence of a magnetic
field, which determines the ability to control the object's movement
within a magnetic field. The μm for an individual particle at
room temperature and above is defined as

μ m = π μ 0 M S , C 2 D C 5 324
k B T η

[0004]where μ0 is the magnetic constant, M.sub.S,C is the
saturation magnetic moment of the mNPs, DC is the diameter of the
mNPs, kB is the Boltzmann constant, η is the viscosity of the
medium, and T is the temperature. Because of their small particle size,
which results in randomized magnetic moments, the μm for mNPs is
usually small. This leads to an intrinsic challenge for applications
where the favorable diffusive properties of the small mNPs are
advantageous, for example, where the mNPs are used to capture diagnostic
targets via antibody-antigen interactions. On the one hand, the small
particles display better association and binding properties, but on the
other hand their small size reduces magnetic capture efficiency.

[0005]Approaches to overcoming the small μm for mNPs include using
larger macromolecules or objects that are labeled with multiple mNPs,
making the mNPs from materials with larger M.sub.S,C using irreversibly
aggregated mNPs, or using high magnetic gradients. However, most of these
approaches result in the loss of favorable diffusive properties and
suffer some drawbacks in the microfluidic-based diagnostic device
environment. High magnetic gradients require a large number of coils and
high current, which cannot be easily integrated into microfluidic
devices. Materials with high M.sub.S,C are usually metals or alloys and,
because of their high surface/volume ratio, they are prone to oxidation
events that can lower their M.sub.S,C. The pre-aggregation of mNPs into
larger structures results in the permanent lowering of surface/volume
ratio and to a decrease in the mNPs favorable diffusive properties. There
is a need for mNPs with favorable diffusive properties that can also be
readily separated in a small magnetic field.

[0006]Stimuli-responsive ("intelligent" or "smart") materials and
molecules exhibit abrupt property changes in response to small changes in
external stimuli such as pH; temperature; UV-visible light; ionic
strength; the concentration of certain chemicals, such as polyvalent
ions, polyions of either charge, or enzyme substrates, such as glucose;
as well as upon photo-irradiation or exposure to an electric field.
Normally these changes are fully reversible once the stimulus has been
removed.

[0007]Poly(N-isopropylacrylamide) (PNIPAAm) is a temperature-responsive
polymer that exhibits a lower critical solution temperature (LCST) around
which the polymer reversibly aggregates. Below the LCST, PNIPAAm chains
hydrate to form an expanded structure; above the LCST, PNIPAAm chains
dehydrate to form a shrinkage structure. This property is due to the
thermally-reversible interaction of water molecules with the hydrophobic
groups, especially the isopropyl groups, leading to low entropy,
hydrophobically-bound water molecules below the LCST and release of those
water molecules at and above the LCST. Modification of mNPs with PNIPAAm
yields particles that can be reversibly aggregated in solution as the
temperature is cycled through the LCST.

[0008]Previous work with PNIPAAm-modified mNPs has relied on
post-synthesis chemical modification of the particles. Chiu et al.
synthesized a Fe3O4 ferrofluid by co-precipitating FeCl3
and FeCl2. The ferrofluid was then mixed with a PNIPAAm solution and
crosslinked to form magnetic polymeric networks. Lin, C. L. and W. Y.
Chiu, J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 5923-5934. Wang et
al. also co-precipitated FeCl3 and FeCl2 to synthesize
Fe3O4 particles. Deng, Y., et al., Adv. Mater. 2003, 15,
1729-1732. The particles were coated with a layer of silica and modified
with 3-aminopropyltrimethoxysilane to seed the precipitation
polymerization of NIPAAm. In both methods, the post-synthesis
functionalization requires multiple steps and can result in particle
aggregation. There is a need for methods of making stimuli-responsive
polymer-modified mNPs that do not require extensive post-synthesis workup
steps and result in minimal particle aggregation.

[0009]Stimuli-responsive materials and molecules have numerous possible
applications in the biomedical/pharmaceutical field, as well as in
biotechnology and the related industries. Smart conjugates, smart
surfaces, smart polymeric micelles, and smart hydrogels have all been
studied for a variety of diagnostics, separations, cell culture, drug
delivery, and bioprocess applications.

[0010]Despite the development of magnetic nanoparticle (mNP) technologies
for diagnostic and imaging applications, there exists a need for a
stimuli-responsive magnetic nanoparticle with favorable diffusive
properties as well as with the ability to be reversibly aggregated into
larger structures, and simpler methods for making the nanoparticles. The
present invention seeks to fulfill these needs and provides further
related advantages.

SUMMARY OF THE INVENTION

[0011]In one aspect, the invention provides a stimuli-responsive magnetic
nanoparticle having responsivity to a magnetic field, comprising:

[0012](a) a core having responsivity to a magnetic field; and

[0013](b) a plurality of stimuli-responsive polymers attached to the core,
wherein the polymer terminates with a functional group capable of
covalent coupling with a capture molecule.

[0033](b) increasing the temperature of the medium to above the lower
critical solution temperature of the nanoparticle to provide thermally
aggregated nanoparticles;

[0034](c) subjecting the thermally aggregated nanoparticles to a magnetic
field to provide magnetically aggregated nanoparticles; and

[0035](d) decreasing the temperature to below the lower critical solution
temperature of the nanoparticle and removing the magnetic field to
regenerate the nanoparticles, wherein the regenerated nanoparticles
further comprise the diagnostic target.

[0036]In one embodiment, the invention provides a method for concentrating
a diagnostic target, comprising:

[0038](b) increasing temperature of the medium to above the lower critical
solution temperature of the nanoparticle to provide thermally aggregated
nanoparticles;

[0039](c) subjecting the thermally aggregated nanoparticles to a magnetic
field to provide magnetically aggregated nanoparticles; and

[0040](d) decreasing the temperature to below the lower critical solution
temperature of the nanoparticle and removing the magnetic field to
regenerate the nanoparticles, wherein the regenerated nanoparticles
further comprise the diagnostic target.

[0041]In the above methods, steps (b) to (d) may be repeated.

[0042]In another aspect, the invention also provides devices for using the
stimuli-responsive nanoparticle.

[0043]In one embodiment, the invention provides a device, comprising

[0044](a) a channel adapted for receiving a flow comprising a plurality of
stimulus-responsive magnetic nanoparticles, wherein the nanoparticle is
reversibly self-associative in response to a stimulus; and

[0045](b) a separation region through which the flow passes, wherein the
separation region is adapted to reversibly apply a stimulus and a
magnetic field to the flow.

[0046]The invention also provides assays for using the stimuli-responsive
nanoparticle.

[0047]In one embodiment, the invention provides an assay for detecting a
diagnostic target, comprising:

[0048](a) contacting the diagnostic target with a plurality of
stimuli-responsive magnetic nanoparticles, wherein each nanoparticle
comprises a capture moiety having affinity toward the diagnostic target;

[0054]The foregoing aspects and many of the attendant advantages of this
invention will become more readily appreciated as the same become better
understood by reference to the following detailed description, when taken
in conjunction with the accompanying drawings.

[0055]FIG. 1 is a schematic illustration of a representative method for
making PNIPAAm magnetic NPs were synthesized by using PNIPAAm micelles as
dimensional confinements: The reversible addition fragmentation chain
transfer (RAFT) synthesized PNIPAAm chains formed micelles in tetraglyme
due to the hydrophobic dodecyl group of the chain transfer agent (CTA)
used in the polymerization that drives micelle assembly. The exposed
chain ends on the micelle periphery (corona) is terminated with a
carboxyl group that can be further functionalized. The PNIPAAm micelles
were loaded with Fe(CO)5 and heated at 190° C. for 5 hours to
provide the γ-Fe2O3 cores.

[0056]FIGS. 2A and 2B illustrate representative PNIPAAm mNPs of the
invention. FIG. 2A is a transmission electron microscope (TEM) image of
the PNIPAAm mNPs. FIG. 2B is a size histogram of PNIPAAm mNPs. The images
were obtained by suspending the mNPs in water and depositing onto a
carbon stabilized formvar-coated copper grid. PNIPAAm was not stained,
only the γ-Fe2O3 (inorganic) portion of the mNPs is
visualized. The γ-Fe2O3 nanoparticles in the core exhibit
a spherical shape with an average size of 4.9±0.9 nm.

[0057]FIG. 3 is an X-ray diffraction (XRD) pattern of representative
PNIPAAm mNPs of the invention (solid) that matches the theoretical
γ-Fe2O3 (dashed) index. The broadened pattern for the
PNIPAAm mNPs reflects their polycrystalline nature.

[0059]FIG. 5A is a schematic illustration of a representative device and
method of the invention showing mNP capture and release. FIG. 5B shows
images of the corresponding micrographs. Capture and release of the
PNIPAAm mNPs (steptavidin:biotin PNIPAAm mNPs, (SA bPNIPAAm mNP)) was
demonstrated in PEGylated PDMS microfluidic channels having a channel
width of 500 μm. The magnetic field was introduced by embedding a
magnet on one side (lower) of the channel. The mNPs were flowed into the
channel (reference numeral 100) having a non-fouling surface (reference
numerals 102 and 104) with heater and magnetic field off (heater off,
magnet off). A mNP solution (4 mg/mL) was injected into the channel with
a constant flow (˜1 μL/min). The mNPs are soluble and free
flowing in the PEGylated channels when the temperature is below the LCST
of the PNIPAAm mNPs (free flowing mNPs, reference numeral 10). As the
mNPs flow into the heated region (heater on, magnet off), the temperature
is above the LCST of the mNPs and the mNPs aggregate, but do not adhere
to the non-fouling, PEGylated channel wall surfaces in the absence of an
applied magnetic field (aggregated mNPs, reference numeral 20). The mNPs
are captured onto the PEGylated channel walls only when the temperature
is raised above the LCST, and the magnetic field is applied (heater on,
magnet on) (captured aggregated mNPs, reference numeral 30). The reversal
of the temperature and applied magnetic field (heater off, magnet off)
results in the release and redispersion of the captured aggregated mNPs
and their diffusive re-entry into the flow stream (free flowing mNPs,
reference numeral 10).

[0060]FIG. 6 is a graph illustrating the capture and release kinetics of
representative mNPs of the invention (bPNIPAAm mNPs) in the PEGylated
channel of FIG. 5. Starting from time zero (square curve), 20 μL of
the mNPs were injected into the channel with a constant flow (˜1
μL/min). The channel reached the maximum capture density within 15
minutes. When the temperature was reversed to below the LCST and the
applied field was removed (time zero of release, circle curve), the
captured particles were quantitatively released back into the flow stream
within 10 minutes.

DETAILED DESCRIPTION OF THE INVENTION

[0061]The present invention provides a stimuli-responsive magnetic
nanoparticle, methods of making the nanoparticles, and methods of using
the nanoparticles.

[0062]Stimuli-responsive magnetic nanoparticles having terminal functional
groups. In one aspect, the invention provides a stimuli-responsive
magnetic nanoparticle having responsivity to a magnetic field,
comprising:

[0063](a) a core having responsivity to a magnetic field; and

[0064](b) a plurality of stimuli-responsive polymers attached to the core,
wherein the polymer terminates with a functional group capable of
covalent coupling with a capture molecule.

[0065]The core includes material having responsivity to magnetic field.
Suitable materials having responsivity to a magnetic field include metal
oxides, such as ferrous oxide, ferric oxide, gadolinium oxide, and
mixtures thereof. Mixtures of one or more metal oxide can be used. In one
embodiment, the core comprises ferric oxide.

[0066]In addition to magnetic materials, the core can include non-magnetic
materials, such as silicon nitride, stainless steel, titanium, and nickel
titanium. Mixtures of one or more non-magnetic materials can also be
used.

[0067]The core of the nanoparticles of the invention has a diameter from
about 2 nm to about 10 nm. In one embodiment, the core has a diameter
from about 4.0 nm to 6.0 nm.

[0068]Stimuli-responsive polymers. The nanoparticles of the invention
include a plurality of stimuli-responsive polymers attached to the core.
In one embodiment, the plurality of stimuli-responsive polymers attached
to the core forms a corona. As used herein, the term "corona" refers to
the sphere or coating of stimuli-responsive polymers surrounding the
core.

[0069]The stimuli-responsive polymer can be any polymer having a
stimuli-responsive property. The stimuli-responsive polymer can be any
one of a variety of polymers that change their associative properties
(e.g., change from hydrophilic to hydrophobic) in response to a stimulus.
The stimuli-responsive polymer responds to changes in external stimuli
such as the pH, temperature, UV-visible light, photo-irradiation,
exposure to an electric field, ionic strength, and the concentration of
certain chemicals by exhibiting property change. The chemicals could be
polyvalent ions such as calcium ion, polyions of either charge, or enzyme
substrates such as glucose. For example, the temperature-responsive
polymer is responsive to changes in temperature by exhibiting a LCST in
aqueous solution. The stimuli-responsive polymer can be a
multi-responsive polymer, where the polymer exhibits property change in
response to combined simultaneous or sequential changes in two or more
external stimuli.

[0070]The stimuli-responsive polymers may be synthetic or natural polymers
that exhibit reversible conformational or physico-chemical changes such
as folding/unfolding transitions, reversible precipitation behavior, or
other conformational changes to in response to stimuli, such as to
changes in temperature, light, pH, ions, or pressure. Representative
stimuli-responsive polymers include temperature-sensitive polymers, a
pH-sensitive polymers, and a light-sensitive polymers.

[0073]The stimuli-responsive polymers useful in the nanoparticles of the
invention include homopolymers and copolymers having stimuli-responsive
behavior. Other suitable stimuli-responsive polymers include block and
graft copolymers having one or more stimuli-responsive polymer
components. A suitable stimuli-responsive block copolymer may include,
for example, a temperature-sensitive polymer block, or a pH-sensitive
block. A suitable stimuli-responsive graft copolymer may include, for
example, a pH-sensitive polymer backbone and pendant
temperature-sensitive polymer components, or a temperature-sensitive
polymer backbone and pendant pH-sensitive polymer components.

[0074]The stimuli-responsive polymer can include a polymer having a
balance of hydrophilic and hydrophobic groups, such as polymers and
copolymers of N-isopropylacrylamide. An appropriate
hydrophilic/hydrophobic balance in a smart vinyl type polymer is
achieved, for example, with a pendant hydrophobic group of about 2-6
carbons that hydrophobically bond with water, and a pendant polar group
such as an amide, acid, amine, or hydroxyl group that H-bond with water.
Other polar groups include sulfonate, sulfate, phosphate and ammonium
ionic groups. Preferred embodiments are for 3-4 carbons (e.g., propyl,
isopropyl, n-butyl, isobutyl, and t-butyl) combined with an amide group
(e.g. PNIPAAm), or 2-4 carbons (e.g., ethyl, propyl, isopropyl, n-butyl,
isobutyl, and t-butyl) combined with a carboxylic acid group (e.g.,
PPAA). There is also a family of smart A-B-A (also A-B-C) block
copolymers of polyethers, such as PLURONIC polymers having compositions
of PEO-PPO-PEO, or polyester-ether compositions such as PLGA-PEG-PLGA. In
one embodiment, the stimuli-responsive polymer is a temperature
responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm).

[0075]The stimuli-responsive polymer useful in the invention can be a
smart polymer having different or multiple stimuli-responsivities, such
as homopolymers responsive to pH or light. Block, graft, or random
copolymers with dual sensitivities, such as pH and temperature, light and
temperature, or pH and light, may also be used.

[0076]The stimuli-responsive polymer can contain a micelle-forming
hydrophobic moiety. The hydrophobic group can be a saturated or
unsaturated alkyl group, a hydrophobic oligomer or polymer such as a
polyester, polyamide or polypeptide, any one of which could be
incorporated as a block in a block copolymer, or a pendant graft polymer
in a graft copolymer. In one embodiment, the micelle-forming hydrophobic
moiety is an alkyl group. In one embodiment, the micelle-forming
hydrophobic moiety is a n-dodecyl group.

[0077]Temperature-Sensitive Polymers. Illustrative embodiments of the many
different types of temperature-sensitive polymers that may be conjugated
to interactive molecules are polymers and copolymers of N-isopropyl
acrylamide (NIPAAm). PolyNIPAAm is a thermally sensitive polymer that
precipitates out of water at 32° C., which is its lower critical
solution temperature (LCST), or cloud point (Heskins and Guillet, J.
Macromol. Sci.-Chem. A2:1441-1455, 1968). When polyNIPAAm is
copolymerized with more hydrophilic comonomers such as acrylamide, the
LCST is raised. The opposite occurs when it is copolymerized with more
hydrophobic comonomers, such as N-t-butyl acrylamide. Copolymers of
NIPAAm with more hydrophilic monomers, such as AAm, have a higher LCST,
and a broader temperature range of precipitation, while copolymers with
more hydrophobic monomers, such as N-t-butyl acrylamide, have a lower
LCST and usually are more likely to retain the sharp transition
characteristic of PNIPAAm (Taylor and Cerankowski, J. Polymer Sci.
13:2551-2570, 1975; Priest et al., ACS Symposium Series 350:255-264,
1987; and Heskins and Guillet, J. Macromol. Sci.-Chem. A2:1441-1455,
1968, the disclosures of which are incorporated herein). Copolymers can
be produced having higher or lower LCSTs and a broader temperature range
of precipitation.

[0078]The synthesis of an amino-terminated polymer proceeds by the radical
polymerization of NIPAAm in the presence of AIBN as an initiator and
1-aminoethanethiol-hydrochloride as a chain transfer reagent. To
synthesize a chain with --COOH or --OH terminal groups, carboxyl- or
hydroxyl-thiol chain transfer agents, respectively, have been used
instead of the amino-thiol. It should be noted that the synthesis of the
end-reactive polymers is based on a chain transfer initiation and
termination mechanism. This yields a relatively short polymer chain,
having a molecular weight somewhere between 1000 and 25,000 to 30,000.
The shortest chains, less than 10,000 in molecular weight, are usually
called "oligomers." Oligomers of different molecular weights can be
synthesized by simply changing the ratio of monomer to chain transfer
reagent, and controlling their concentration levels, along with that of
the initiator. The polymers useful in the invention may also be prepared
by reversible addition fragmentation chain transfer (RAFT)
polymerization.

[0079]Oligomers of NIPAAm (or other vinyl monomers) having a reactive
group at one end are prepared by the radical polymerization of NIPAAm
using AIBN as initiator, plus a chain transfer agent with a thiol (--SH)
group at one end and the desired "reactive" group (e.g., --OH, --COOH,
--NH2) at the other end. Chen and Hoffman, Bioconjugate Chem.
4:509-514, 1993 and Chen and Hoffman, J. Biomaterials Sci. Polymer Ed.
5:371-382, 1994, each of which is incorporated herein by reference.
Appropriate quantities of NIPAAm, AIBN and chain transfer reagent in DMF
are placed in a thick-walled polymerization tube and the mixtures are
degassed by freezing and evacuating and then thawing (4 times). After
cooling for the last time, the tubes are evacuated and sealed prior to
polymerization. The tubes are immersed in a water bath at 60° C.
for 4 h. The resulting polymer is isolated by precipitation into diethyl
ether and weighed to determine yield. The molecular weight of the polymer
is determined either by titration (if the end group is amine or
carboxyl), by vapor phase osmometry (VPO), or gel permeation
chromatography (GPC).

[0080]Temperature sensitive oligopeptides also may be incorporated into
the nanoparticles.

[0081]pH-Sensitive Polymers. Synthetic pH-sensitive polymers useful in
making the nanoparticles described herein are typically based on
pH-sensitive vinyl monomers, such as acrylic acid (AAc), methacrylic acid
(MAAc) and other alkyl-substituted acrylic acids such as ethylacrylic
acid (EAAc), propylacrylic acid (PAAc), and butylacrylic acid (BAAc),
maleic anhydride (MAnh), maleic acid (MAc), AMPS
(2-acrylamido-2-methyl-1-propanesulfonic acid), N-vinyl formamide (NVA),
N-vinyl acetamide (NVA) (the last two may be hydrolyzed to polyvinylamine
after polymerization), aminoethyl methacrylate (AEMA), phosphoryl ethyl
acrylate (PEA) or methacrylate (PEMA). pH-Sensitive polymers may also be
synthesized as polypeptides from amino acids (e.g., polylysine or
polyglutamic acid) or derived from naturally-occurring polymers such as
proteins (e.g., lysozyme, albumin, casein), or polysaccharides (e.g.,
alginic acid, hyaluronic acid, carrageenan, chitosan, carboxymethyl
cellulose) or nucleic acids, such as DNA. pH-Responsive polymers usually
contain pendant pH-sensitive groups such as --OPO(OH)2, --COOH, or
--NH2 groups. With pH-responsive polymers, small changes in pH can
stimulate phase-separation, similar to the effect of temperature on
solutions of PNIPAAm (Fujimura et al. Biotech. Bioeng. 29:747-752
(1987)). By randomly copolymerizing a thermally-sensitive NIPAAm with a
small amount (e.g., less than 10 mole percent) of a pH-sensitive
comonomer such as AAc, a copolymer will display both temperature and pH
sensitivity. Its LCST will be almost unaffected, sometimes even lowered a
few degrees, at pHs where the comonomer is not ionized, but it will be
dramatically raised if the pH-sensitive groups are ionized. When the
pH-sensitive monomer is present in a higher content, the LCST response of
the temperature sensitive component may be "eliminated" (e.g., no phase
separation seen up to and above 100° C.).

[0082]Graft and block copolymers of pH and temperature sensitive monomers
can be synthesized that retain both pH and temperature transitions
independently. Chen, G. H., and A. S. Hoffman, Nature 373:49-52, 1995.
For example, a block copolymer having a pH-sensitive block (polyacrylic
acid) and a temperature sensitive block (PNIPAAm) can be useful in the
invention.

[0083]Light-Sensitive Polymers. Light-responsive polymers usually contain
chromophoric groups pendant to or along the main chain of the polymer
and, when exposed to an appropriate wavelength of light, can be
isomerized from the trans to the cis form, which is dipolar and more
hydrophilic and can cause reversible polymer conformational changes.
Other light sensitive compounds can also be converted by light
stimulation from a relatively non-polar hydrophobic, non-ionized state to
a hydrophilic, ionic state.

[0084]In the case of pendant light-sensitive group polymers, the
light-sensitive dye, such as aromatic azo compounds or stilbene
derivatives, may be conjugated to a reactive monomer (an exception is a
dye such as chlorophyllin, which already has a vinyl group) and then
homopolymerized or copolymerized with other conventional monomers, or
copolymerized with temperature-sensitive or pH-sensitive monomers using
the chain transfer polymerization as described above. The light sensitive
group may also be conjugated to one end of a different (e.g.,
temperature) responsive polymer. A number of protocols for such
dye-conjugated monomer syntheses are known.

[0085]Although both pendant and main chain light sensitive polymers may be
synthesized and are useful for the methods and applications described
herein, the preferred light-sensitive polymers and copolymers thereof are
typically synthesized from vinyl monomers that contain light-sensitive
pendant groups. Copolymers of these types of monomers are prepared with
"normal" water-soluble comonomers such as acrylamide, and also with
temperature- or pH-sensitive comonomers such as NIPAAm or AAc.

[0086]Light-sensitive compounds may be dye molecules that isomerize or
become ionized when they absorb certain wavelengths of light, converting
them from hydrophobic to hydrophilic conformations, or they may be other
dye molecules which give off heat when they absorb certain wavelengths of
light. In the former case, the isomerization alone can cause chain
expansion or collapse, while in the latter case the polymer will
precipitate only if it is also temperature-sensitive.

[0087]Light-responsive polymers usually contain chromophoric groups
pendant to the main chain of the polymer. Typical chromophoric groups
that have been used are the aromatic diazo dyes (Ciardelli, Biopolymers
23:1423-1437, 1984; Kungwatchakun and Irie, Makromol. Chem., Rapid
Commun. 9:243-246, 1988; Lohmann and Petrak, CRC Crit. Rev. Therap. Drug
Carrier Systems 5:263, 1989; Mamada et al., Macromolecules 23:1517, 1990,
each of which is incorporated herein by reference). When this type of dye
is exposed to 350-410 nm UV light, the trans form of the aromatic diazo
dye, which is more hydrophobic, is isomerized to the cis form, which is
dipolar and more hydrophilic, and this can cause polymer conformational
changes, causing a turbid polymer solution to clear, depending on the
degree of dye-conjugation to the backbone and the water solubility of the
main unit of the backbone. Exposure to about 750 nm visible light will
reverse the phenomenon. Such light-sensitive dyes may also be
incorporated along the main chain of the backbone, such that the
conformational changes due to light-induced isomerization of the dye will
cause polymer chain conformational changes. Conversion of the pendant dye
to a hydrophilic or hydrophobic state can also cause individual chains to
expand or contract their conformations. When the polymer main chain
contains light sensitive groups (e.g., azo benzene dye) the
light-stimulated state may actually contract and become more hydrophilic
upon light-induced isomerization. The light-sensitive polymers can
include polymers having pendant or backbone azobenzene groups.

[0088]Specific Ion-Sensitive Polymers. Polysaccharides, such as
carrageenan, that change their conformation, for example, from a random
to an ordered conformation, as a function of exposure to specific ions,
such as potassium or calcium, can also be used as the stimulus-responsive
polymers. In another example, a solution of sodium alginate may be gelled
by exposure to calcium. Other specific ion-sensitive polymers include
polymers with pendant ion chelating groups, such as histidine or EDTA.

[0089]Dual- or Multi-Sensitivity Polymers. If a light-sensitive polymer is
also thermally-sensitive, the UV- or visible light-stimulated conversion
of a chromophore conjugated along the backbone to a more hydrophobic or
hydrophilic conformation can also stimulate the dissolution or
precipitation of the copolymer, depending on the polymer composition and
the temperature. If the dye absorbs the light and converts it to thermal
energies rather than stimulating isomerization, then the localized
heating can also stimulate a phase change in a temperature-sensitive
polymer such as PNIPAAm, when the system temperature is near the phase
separation temperature. The ability to incorporate multiple
sensitivities, such as temperature and light sensitivity, or temperature
and pH sensitivity, along one backbone by vinyl monomer copolymerization
lends great versatility to the synthesis and properties of the responsive
polymer-protein conjugates. For example, dyes can be used which bind to
protein recognition sites, and light-induced isomerization can cause
loosening or detachment of the dye from the binding pocket (Bieth et al.,
Proc. Natl. Acad. Sci. USA 64:1103-1106, 1969). This can be used for
manipulating affinity processes by conjugating the dye to the free end of
a temperature responsive polymer, such as ethylene oxide-propylene oxide
(EO-PO) random copolymers available from Carbide. These polymers,
--(CH2CH2O)x--(CH2CHCH3O)y--, have two
reactive end groups. The phase separation point (cloud point) can be
varied over a wide range, depending on the EO/PO ratio, molecular weight,
and concentration, and one end may be derivatized with the ligand dye and
the other end with an --SH reactive group, such as vinyl sulfone (VS).

[0090]Binding Pairs. In one embodiment, the stimuli-responsive magnetic
particles include polymers having terminal functional groups for
covalently coupling a capture molecule. The terminal functional group on
the stimuli-responsive polymer refers to any reactable group that may be
derivatized to make it reactive with the capture moiety, such as
carboxyl, hydroxyl, and amine groups. The terminal functional group may
be derivatized to form reactive groups such as thiol, ketone, N-hydroxy
succinimide esters, N-hydroxy maleimide esters, carbonyl imidazoles,
carbodiimide esters, vinyl sulfone, acrylate, benzyl halide, tosylate,
tresylate, aldehyde, hydrazone, acid halide, p-nitrophenolic esters, and
hydroperoxides. In one embodiment, the terminal functional group on the
stimuli-responsive polymer is a carboxylic group.

[0091]The terminal functional group on the stimuli-responsive polymer can
be coupled with a capture molecule through covalent bonds, including but
not limited to amide, esters, ether, thioether, disulfide, hydrazide,
hydrazone, acetal, ketal, ketone, anhydride, urethane, urea, and
carbamate bonds. In one embodiment, the biotin moiety is coupled to the
stimuli-responsive polymer through an amide bond.

[0092]The terminal functional group can be covalently coupled to a capture
molecule, such as a protein, a nucleic acid oligomer (DNA or RNA), an
antibody, an antigen, an enzyme or an enzyme substrate. The capture
moiety can be further coupled with a target molecule, such as a protein,
a nucleic acid oligomer (DNA or RNA), an antigen, an antibody, an enzyme,
or an enzyme substrate through covalent or non-covalent interaction. In
one embodiment, the terminal functional group is coupled to a biotin, the
capture molecule, to afford a biotinylated nanoparticle. In one
embodiment, the biotinylated nanoparticle can be further conjugated to a
streptavidin, the target molecule, to yield a streptavidin-conjugated
biotinylated nanoparticle, that can be coupled to a biotinylated target
molecule.

[0093]A capture molecule and a target molecule form a binding pair. Each
has an affinity toward the other (e.g., antigen and antibody). Each of
the capture molecule and the target molecule can be a variety of
different molecules, including peptides, proteins, poly- or
oligosaccharides, glycoproteins, lipids and lipoproteins, and nucleic
acids, as well as synthetic organic or inorganic molecules having a
defined bioactivity, such as an antibiotic or anti-inflammatory agent,
that binds to a target site, such as a cell membrane receptor. The
exemplary proteins include antibodies (monoclonal, polyclonal, chimeric,
single-chain or other recombinant forms), their protein/peptide antigens,
protein/peptide hormones, streptavidin, avidin, protein A, protein G,
growth factors and their respective receptors, DNA-binding proteins, cell
membrane receptors, endosomal membrane receptors, nuclear membrane
receptors, neuron receptors, visual receptors, and muscle cell receptors.
Exemplary oligonucleotides include DNA (genomic or cDNA), RNA, antisense,
ribozymes, and external guide sequences for RNAase P, and can range in
size from short oligonucleotide primers up to entire genes. Carbohydrates
include tumor associated carbohydrates (e.g., Lex, sialyl Lex,
Ley, and others identified as tumor associated as described in U.S.
Pat. No. 4,971,905, incorporated herein by reference), carbohydrates
associated with cell adhesion receptors (e.g., Phillips et al., Science
250:1130-1132, 1990), and other specific carbohydrate binding molecules
and mimetics thereof which are specific for cell membrane receptors.

[0094]Among the proteins, streptavidin is particularly useful as a model
for other capture moiety-target molecule binding pair systems described
herein. Streptavidin is an important component in many separations and
diagnostic technologies which use the very strong association of the
streptavidin-biotin affinity complex. (Wilchek and Bayer, Avidin-Biotin
Technology, New York, Academic Press, Inc., 1990; and Green, Meth.
Enzymol. 184:51-67. Protein G, a protein that binds IgG antibodies
(Achari et al., Biochemistry 31:10449-10457, 1992, and Akerstrom and
Bjorck, J. Biol. Chem. 261:10240-10247, 1986) is also useful as a model
system. Representative immunoaffinity molecules include engineered single
chain Fv antibody (Bird et al., Science 242:423-426, 1988 and U.S. Pat.
No. 4,946,778 to Ladner et al., incorporated herein by reference, Fab,
Fab', and monoclonal or polyclonal antibodies.

[0095]In one embodiment, the capture molecule is an antibody and the
target molecule is an antigen. In another embodiment, both the capture
molecule and the target molecule are protein. In another embodiment, the
capture molecule is a nucleic acid (DNA or RNA) and the target molecule
is a complimentary nucleic acid (DNA or RNA). In another embodiment, the
target molecule is a nucleic acid (DNA or RNA) and the capture molecule
is a protein. In another embodiment, the capture molecule is a cell
membrane receptors and the target molecule is a ligand. In another
embodiment, the capture moiety is an enzyme and the target molecule is a
substrate. In another embodiment, the capture molecule is biotin and the
target molecule is streptavidin or avidin. In another embodiment, the
target moiety is a cell (e.g., a living cell).

[0098](b) a plurality of stimuli-responsive polymers attached to the core,
wherein the polymer terminates with a capture moiety.

[0099]In one embodiment, the capture moiety is an antibody. In one
embodiment, the capture moiety is an antigen. In one embodiment, the
capture moiety is a nucleic acid oligomer (DNA or RNA). In one
embodiment, the capture moiety is an enzyme substrate. In one embodiment,
the capture moiety is biotin.

[0100]In one embodiment, the capture moiety is a biotin or biotin
derivative having affinity to avidin or streptavidin. In this embodiment,
the nanoparticle includes a plurality of biotin moieties coupled to the
stimuli-responsive polymer. In one embodiment, the biotin or derivative
is coupled to the polymer through an amide bond. In one embodiment, the
nanoparticles of the invention have from about 50 to about 100 biotin
moieties/nanoparticle. In one embodiment, the nanoparticle is further
coupled to streptavidin molecules via biotin moieties. In one embodiment,
the nanoparticle has from about 30 to about 70
streptavidins/nanoparticle.

[0101]Nanoparticle properties. The nanoparticles of the invention may have
diameters of from about 3 nm to about 70 nm. In one embodiment, the
nanoparticles have diameters from about 5 nm to about 30 nm. In one
embodiment, the nanoparticles have diameters from about 5 nm to about 10
nm. In one embodiment, the nanoparticles have diameters from about 4 nm
to about 6 nm.

[0103]The characterization of representative stimuli-responsive mNPs,
PNIPAAm mNPs, were carried out by transmission electron microscopy (TEM)
and dynamic light scattering (DLS) analysis. The TEM images and resulting
size histograms are shown in FIGS. 2A and 2B, respectively. Only the
γ-Fe2O3 (inorganic) portion of the mNPs was visualized,
the PNIPAAm is not stained. The inorganic portion of the particles
exhibits a spherical shape with an average size of 4.9±0.9 nm. The
number averaged particle diameter from a multimodal DLS size distribution
was 6.7±2.7 nm for the PNIPAAm mNP, and 11.5±1.7 nm for the
bPNIPAAm mNP. The crystal structure of mNPs was characterized by X-ray
diffraction (XRD) analysis (FIG. 3). The XRD pattern of the PNIPAAm mNPs
(solid) matches the theoretical γ-Fe2O3(dashed) spectrum.
The broadened pattern for the PNIPAAm mNPs reflects their polycrystalline
nature.

[0104]The magnetic properties of the exemplary temperature responsive
mNPs, PNIPAAm mNP, were characterized with a Superconducting Quantum
Interference Device (SQUID) at a field range of ±5 T (field vs.
magnetization, H-M) and a temperature range from 5 to 300 K (temperature
vs. magnetization, T-M). H-M measurements (FIG. 4A) were used to
ascertain the induced magnetization from the nanoparticles at the applied
field. Magnetization values at a 5 T applied field were 8 and 11 emu/g
for room temperature and 5 K, respectively. While the room temperature
H-M measurement displays almost no hysteresis, the same measurement at 5
K (FIG. 4aB) shows a coercivity of 450 Oe. The T-M measurement (FIG. 4C)
shows a blocking temperature, TB, of 25 K. Zero-field-cooled (ZFC)
and field-cooled (FC) curves overlap above TB, which can be
correlated to the size distribution. The results of the SQUID
measurements confirm these PNIPAAm modified mNPs are superparamagnetic.

[0105]Methods for making stimuli-responsive nanoparticles. In another
aspect, the invention provides a method for making stimuli-responsive
nanoparticles.

[0106]In one embodiment, the stimuli-responsive nanoparticles are made by
the following steps:

[0107](a) providing a plurality of stimuli-responsive polymers to form a
micelle having a hydrophobic core; and

[0108](b) loading the hydrophobic core with material having responsivity
to a magnetic field.

[0109]In the above method, the hydrophobic core formed by the micelle can
be used as dimensional confinement to synthesize a core having
responsivity to a magnetic field.

[0110]In one embodiment, the stimuli-responsive mNPs are
temperature-responsive mNPs. The temperature-responsive mNPs can be
synthesized from temperature-responsive polymeric micelles. In one
embodiment, telechelic poly(N-isopropylacrylamide) (PNIPAAm) polymer
chains were synthesized with dodecyl tails at one end and a reactive
carboxylate at the opposite end by the reversible addition fragmentation
chain transfer (RAFT) technique. These PNIPAAm chains self-associate into
micelles that were used as dimensional confinements to synthesize the
magnetic nanoparticles. The resulting superparamagnetic nanoparticles
exhibit a γ-Fe2O3 core (˜5 nm) with a layer of
carboxylate-terminated PNIPAAm on the surface. The carboxylate-group was
used to functionalize the magnetic nanoparticles with biotin and
subsequently streptavidin.

[0111]The PNIPAAm mNPs described above were synthesized in one-step with a
polymeric micelle approach that was utilized to take advantage of the
RAFT synthesis technique (FIG. 1). The PNIPAAm chains were synthesized
from a RAFT chain transfer agent (CTA) that contains a hydrophobic
dodecyl group at one end and a carboxyl group at the other end. These
chains formed micelles in tetraglyme solvent, driven by the association
of the core-forming dodecyl groups. The polydispersity index of the
RAFT-synthesized PNIPAAm was less than 1.10, which correspondingly
yielded micelles with narrow size dispersities (hydrodynamic diameter of
27 nm with 13.5 nm half-widths at maximum intensity). The PNIPAAm
micelles were loaded with the iron oxide reactants, where the micelles
served as dimensional confinements for the synthesis of thermal
responsive iron oxide mNPs. The telechelic nature of the RAFT-synthesized
chain ends in the exposed PNIPAAm coating layer could be readily
exploited to subsequently conjugate biotin groups via the solvent-exposed
end carboxyl groups. The biotinylated particles (bPNIPAAm mNPs) were
complexed with streptavidin (SA) as SA-bPNIPAAm mNPs. The HABA assay was
used to quantify the number of biotins and SA, with a result of 89
biotins per bPNIPAAm mNP and 46 SA per bPNIPAAm mNP.

[0112]Methods for using the stimuli-responsive nanoparticles. In other
aspects, the invention provides methods for using the nanoparticle.

[0113]In one embodiment, the invention provides a method for capturing a
diagnostic target, comprising:

[0116](c) subjecting the aggregated nanoparticle to a magnetic field to
provide magnetically aggregated nanoparticles; and

[0117](d) removing the stimulus and the magnetic field to regenerate the
nanoparticles, wherein the regenerated nanoparticles further comprise the
diagnostic target.

[0118]In the above methods, the external stimulus could be temperature,
pH, or light. In one embodiment the stimuli-responsive magnetic
nanoparticle is a pH-responsive nanoparticle, and the external stimulus
is the pH. In one embodiment the stimuli-responsive magnetic nanoparticle
is a light-responsive nanoparticle, and the external stimulus is light.
In one embodiment the stimuli-responsive magnetic nanoparticle is
ion-responsive nanoparticle, and the external stimulus is the ion
strength of a specific ion.

[0119]In one embodiment, the stimulus is temperature. The invention
provides a method for capturing a diagnostic target, comprising:

[0121](b) increasing the temperature of the medium to above the lower
critical solution temperature of the nanoparticle to provide thermally
aggregated nanoparticles;

[0122](c) subjecting the thermally aggregated nanoparticles to a magnetic
field to provide magnetically aggregated nanoparticles; and

[0123](d) decreasing the temperature to below the lower critical solution
temperature of the nanoparticle and removing the magnetic field to
regenerate the nanoparticles, wherein the regenerated nanoparticles
further comprise the diagnostic target.

[0124]In one embodiment, the invention provides a method for concentrating
a diagnostic target, comprising:

[0126](b) increasing temperature of the medium to above the lower critical
solution temperature of the nanoparticle to provide thermally aggregated
nanoparticles;

[0127](c) subjecting the thermally aggregated nanoparticles to a magnetic
field to provide magnetically aggregated nanoparticles; and

[0128](d) decreasing the temperature to below the lower critical solution
temperature of the nanoparticle and removing the magnetic field to
regenerate the nanoparticles, wherein the regenerated nanoparticles
further comprise the diagnostic target.

[0129]In the above methods, steps (b) to (d) may be repeated.

[0130]In the above methods, the diagnostic target molecule and the capture
moiety each has affinity toward the other and are capable of forming a
binding pair. As used herein, the term "diagnostic target" refers to a
molecule that is indicative of a diseased condition or an indicator of
exposure to a toxin, or a therapeutic drug that has been administered to
a subject and whose concentration is to be monitored.

[0131]In one embodiment, the diagnostic target molecule is an antibody and
the capture moiety is an antigen. In one embodiment, the diagnostic
target molecule is an antigen and the capture moiety is an antibody. In
one embodiment, the diagnostic target molecule is a nucleic acid oligomer
(RNA or DNA) and the capture moiety is a complementary nucleic acid
oligomer. In one embodiment, the diagnostic target molecule is a nucleic
acid oligomer (RNA or DNA) and the capture moiety is a protein. In one
embodiment, the diagnostic target molecule is a protein and the capture
moiety is a nucleic acid oligomer (RNA or DNA). In one embodiment, the
diagnostic target molecule is an enzyme and the capture moiety is a
substrate. In one embodiment, the diagnostic target molecule is an enzyme
substrate and the capture moiety is an enzyme.

[0132]In one embodiment, the methods can be carried out in point-of-care
microfluidic devices. In one embodiment, the methods can be carried out
in the microfluidic channel settings. In one embodiment, the methods can
be carried out in microfluidic lab card settings.

[0133]The dual magneto- and thermally-responsive mNPs of the invention are
designed to facilitate diagnostic target isolation and/or assay. The
temperature responsive mNPs reversibly aggregate as the temperature is
cycled above and below the LCST. Aggregation of the mNPs results in an
increase of the effective particle size, facilitating the magnetic
separation of the particles to the channels walls out of the flow stream
with a small applied field. As the temperature is reversed below the LCST
and the applied field is removed, the captured particles can be recovered
quickly by re-entry into the flow stream.

[0134]A scheme for the representative PNIPAAm mNP separation system is
shown in FIG. 5A. The mNPs are soluble and free flowing in the PEGylated
channels (channel surfaces to which have been attached PEG-containing
polymers) when the temperature was held below the LCST of the mNPs. The
size of these PNIPAAm mNPs also gives them low magnetophoretic mobility,
so that they are not captured by an applied magnetic field under flow
conditions below the LCST. The mNPs can thus diffuse and capture targets
as isolated particles below the LCST. As they flow into the heated region
of a microchannel, the temperature is raised above the LCST of the
PNIPAAm and the mNPs aggregate, but do not stick to the non-fouling,
PEGylated channel walls in the absence of an applied magnetic field. The
mNPs are captured onto the PEGylated channel walls only when the
temperature is raised above the LCST, and the magnetic field is applied.
The reversal of the temperature and applied magnetic field results in the
redispersion of the aggregated mNPs and their diffusive re-entry into the
flow stream.

[0135]The LCST was determined by cloud point measurements and found to be
32.4±0.1° C. for the PNIPAAm mNPs, 31.0±0.1° C. for
bPNIPAAm mNPs, and 41.1±0.3° C. for SA-bPNIPAAm mNPs. The
particle capture/release was demonstrated in PEGylated PDMS microfluidic
channels with bPNIPAAm mNPs. The width of the channel was 500 μm. The
magnetic field was introduced by embedding a magnet at the lower side of
the channel. The mNP solution (4 mg/mL) was injected into the channels
with a constant flow (˜1 μL/min). After the injection of
particle solution, the channel was maintained at the same temperature
with the applied field and washed with buffer at the same flow rate.

[0136]FIG. 5B shows the micrographs of the capture/release illustrated
schematically in FIG. 5A. When the temperature was lower than the LCST,
neither aggregation nor capture occurred. Once the temperature was raised
above the LCST, the particles aggregated. When the temperature was higher
than LCST and the applied field was on, the mNPs were captured because
the aggregation results in an increase of DC and μm. For the
release study, buffer was injected (˜1 μL/min) when the
temperature was lower than the LCST and at zero applied field. The
kinetic profile (FIG. 6) of the capture and release was determined by
analyzing the capture micrographs. Starting from time zero, 20 μL of
the mNPs were injected into the channel with a constant flow (˜1
μL/min). The channel reached the maximum capture density within 15
minutes at this flow rate and mNP concentration. After the capture at the
heated wall position, the channel was washed with 20 μL of buffer at
the same flow rate. The captured mNPs are held stable during buffer
washing. When the temperature was reversed to below the LCST and the
applied field was removed (time zero of release), the captured particles
were quantitatively released back into the flow stream within 10 minutes.

[0137]This representative system demonstrated the reversible
magnetophoretic capture of the modified mNPs in PEGylated microfluidic
channels as the DC and μm are increased in the aggregated
state. By reversibly aggregating the particles at a controlled time point
and channel position after the isolated mNP has reacted with target
molecules, the advantages of a large surface/volume ratio and faster
diffusion during target capture are retained, while optimizing μm
for magnetic isolation after target capture.

[0138]Devices that utilize stimuli-responsive nanoparticles. In one
aspect, the invention also provides devices for using the
stimuli-responsive nanoparticle.

[0139]In one embodiment, the invention provides a device, comprising

[0140](a) a channel adapted for receiving a flow comprising a plurality of
stimulus-responsive magnetic nanoparticles, wherein the nanoparticle is
reversibly self-associative in response to a stimulus; and

[0141](b) a separation region through which the flow passes, wherein the
separation region is adapted to reversibly apply a stimulus and a
magnetic field to the flow to capture the nanoparticles.

[0142]In one embodiment, the device is a well of a multi-well plate. In
one embodiment, the device is a microfluidic device having a channel. In
one embodiment, the device's channel further comprises a surface having
an array or plurality of capture regions. As used herein, the term
"capture region" refers to a region of the surface of the channel coated
with a plurality of stimulus-responsive polymers for capturing the
nanoparticles. In one embodiment, the separation region is non-fouling. A
representative device is illustrated in FIG. 5A.

[0143]The device useful in the invention permits reversible,
stimuli-induced aggregation of the stimuli-responsive magnetic
nanoparticles followed by magnetic field-induced aggregation of the
stimuli-induced aggregates.

[0144]Assays that utilize stimuli-responsive nanoparticles. The invention
also provides assays for using the stimuli-responsive nanoparticle.

[0145]In one embodiment, the invention provides an assay for detecting a
diagnostic target, comprising:

[0146](a) contacting the diagnostic target with a plurality of
stimuli-responsive magnetic nanoparticles, wherein each nanoparticle
comprises a capture moiety having affinity toward the diagnostic target;

[0152]In the above method, forming nanoparticle conjugates by combining
the diagnostic target with the stimuli-responsive magnetic nanoparticles
provides a conjugate that includes a diagnostic target bound to the
capture moiety. In the above method, regenerating the nanoparticle
conjugates by removing the stimulus and the magnetic field provides
released, free flowing nanoparticle conjugates in which the diagnostic
target is bound to the capture moiety.

[0153]The regenerated nanoparticles including the diagnostic target can be
analyzed with or without release of the diagnostic target from the
nanoparticle.

[0154]The diagnostic target can be a molecule that is indicative of a
diseased condition or an indicator of exposure to a toxin, or a
therapeutic drug that has been administered to a subject and whose
concentration is to be monitored. The diagnostic target can be any
protein, antibody, or nucleic acid related to a disease. In one
embodiment, the diagnostic target is an antibody against hepatitis B
virus. In one embodiment, the diagnostic target is an antibody against
hepatitis C virus. In one embodiment, the diagnostic target molecule is
an antibody against AIDS virus. In one embodiment, the diagnostic target
molecule is the malaria parasitic antigen, or the antiplasmodial
antibodies, or the parasitic metabolic products, or the plasmodia nucleic
acid fragments. In one embodiment, the diagnostic target molecule is an
antibody against tuberculosis bacteria. In one embodiment, the diagnosis
target molecule is a dengue fever virus or antibody.

[0155]The following examples are provided for the purpose of illustrating,
not limiting, the invention.

EXAMPLES

Example 1

The Preparation and Characterization of Representative
Temperature-Responsive Nanoparticles

[0156]In this example, the preparation and characterization of
representative temperature-responsive nanoparticles of the invention is
described.

[0158]Synthesis of PNIPAAm. Polymerization of PNIPAAm was performed
according to a previously published protocol. Ebara, M., et al., Lab on
Chip 6:843,2006. The target molecular weight was 5,000. Briefly, 2 g of
NIPAAm (monomer), 145 mg of DMP (trithiocarbonate-based chain transfer
agent, CTA), and 6 mg of 4,4'-azobis(4-cyano-valeric acid) (initiator)
were mixed with 5 mL of methanol. After purging with nitrogen for 20 min,
this solution was sealed and maintained at 60° C. overnight. The
methanol was removed by purging air. The polymer was dissolved with THF
and precipitated in pentane for purification. The sample was dried
overnight in a vacuum oven .

[0159]Synthesis of PNIPAAm mNPs. In a typical synthesis, 900 mg (0.18
mmol) of the polymer surfactant (M.W. ˜5,000) was added to 50 mL of
tetraglyme (preheated to 100° C.) and stirred for 5 min.
Subsequently, 0.2 mL of Fe(CO)5 (1.52 mmol) was injected into the
solution and the temperature was raised to 190° C. after 10 min
stirring. The solution was refluxed for 5 hours, and then cooled down to
room temperature. The product was precipitated in n-hexane and collected
by centrifugation. The precipitate was redissolved in deionized water and
dialyzed using a dialysis membrane of MW cut-off of 10,000 for 72 hrs.
After the dialysis, the particles were collected by lyophilization.

[0160]Biotinylation of PNIPAAm mNPs (bPNIPAAm mNPs). PNIPAAm mNPs were
biotinylated via the carboxyl end group with carbodiimide chemistry. The
end carboxyl groups were activated with DCC in the presence of NHS. The
ratio of carboxyl group (PNIPAAm mNPs) to NHS to DCC was 1:1:1. PNIPAAm
mNPs were dissolved in dioxane. The calculated amount of NHS/DCC
solution, which was prepared by premixing NHS and DCC in dioxane, was
slowly added (over 15 minutes) into the particle solution at 12°
C. The mixed solution was stirred overnight and filtered to remove the
urea. The desired amount of EZ-Link® biotin-LC-PEO-amine was
predissolved in dioxane and added to the particle solution, which was
then stirred overnight. The resulting solution was centrifuged to remove
solids. The particles were precipitated with n-hexane followed by
centrifugation and vacuum dried overnight. The particles were dialyzed
against water with a dialysis membrane of MW cut-off of 10,000 for 72 hrs
and collected by lyophilization.

[0162]Lower Critical Solution Temperature (LCST) Measurement. The LCST was
determined as the temperature at 50% of the maximum absorbance at 550 nm.
The concentration of samples was 2 mg/mL in PBS. The data were collected
using a UV-Vis spectrophotometer with a jacketed cuvette holder to
control the temperature of the sample. A heating rate of 0.5°
C./min was used, and absorbance values were measured every
0.5-1.0° C.

[0163]2-(4-Hydroxyphenylazo)benzoic acid (HABA) assay. The biotinylation
efficiency and SA loading were characterized with HABA assay, which used
the HABA/avidin reagent from Sigma. A biotin solution (0.21 mg/mL) was
used for the calibration. The concentration of the particles is 4 mg/mL
in deionized water. The data were collected using a UV-Vis
spectrophotometer. The cuvette with water was used as a blank. After the
water was removed, 450 μL HABA/avidin reagent (reconstituted in
deionized water) into were pipetted into the cuvette. The absorbance at
500 nm was recorded. 10 μL samples were added, mixed by inversion, and
the absorbance recorded at 500 nm. Addition of the sample was repeated
until the total volume of the sample was 50 μL. Because of the
absorbance from the iron oxide core, PNIPAAm mNPs were used as the
control.

[0165]Powder X-Ray Diffraction (XRD). Powder XRD was performed by using a
Philips X-ray diffractometer 1820 (Cu K.sub.αradiation,
γ=1.5418 Å). Two-dimensional patterns were angle integrated to
obtain the patterns displayed. The instrument resolution is 0.02°
in 2θ, and the accumulation time for each sample was 41 minutes.
The 2θ range used was from 20° to 70°. XRD samples
were prepared by coating with several drops of nitrocellulose in amyl
acetate (concentration 1%) on a quartz plate.

[0166]Magnetic Measurements. Magnetic data of the solid samples were
collected with a Quantum Design SQUID MPMS-XL (DC modes and maximum
static field of ±5 T) in liquid helium and room temperatures. The
temperature dependence of the magnetization was measured in the range
5-300 K in an applied field of 20 Oe, after cooling in zero magnetic
field (ZFC) or by cooling in a field of 20 Oe (FC).

Example 2

The Preparation of a Stimuli-Responsive Microfluidic Separation System

[0167]SU-8 Master and PDMS Device Fabrication. A silicon wafer was
spin-coated with SU8-50 and baked at 95° C. for 1 h. The
photoresist was exposed to UV light (Kaspar-Quintel model 2001 aligner)
for 150 s through transparency masks. After exposure, the masters were
baked at 95° C. for 10 min and developed with SU-8 developer
(Microchem) for 15 min. The masters used in this study were 0.1 mm tall
and 0.5 mm wide. Patterned masters and bare silicon wafers were
passivated by 10 minute exposure to silane under vacuum. PDMS prepolymer
was prepared by mixing PDMS base with a curing agent in a 10:1 ratio by
weight and degassing the mixture under vacuum. To fabricate fluid
channels, the mixture was cast against the pattered silicon master. To
fabricate the lower surface, the mixture was cast over a bare silicon
wafer. In both cases the samples were cured at 60° C. for 3 h. A
piece of silicone tubing was embedded into the PDMS to create access
inlets to the channels. PDMS devices were then assembled using O2
plasma bonding.

[0168]ITO Heater Fabrication. ITO heaters were patterned
photolithographically by spin coating AZ 1512 onto ITO slides (Delta
Technologies, Inc.) and exposing to UV through transparency photomasks.
ITO etching was performed by immersing photoresist patterned slides for
7.5 minutes in HNO3:HCl:H2O (1:4:15 by volume) warmed to
55° C. To fabricate electrical conductors, patterned ITO slides
were then coated by 20 nm of chromium followed by 150 nm of gold by
electron beam evaporation. Conductors were patterned
photolithographically as described above. Gold and chromium were etched
by 5 minutes exposure to TFA gold etchant followed by 5 minutes exposure
to TFD chromium etchant. Copper wires were attached to the heater
conductors using silver conductive epoxy.

[0169]UV-induced Graft Polymerization. UV-mediated grafting was directed
according to a previously published protocol. Briefly, benzophenone, a
photosensitizer was dissolved in acetone and flowed into the channel for
less than one minute. The channel was then washed extensively with water.
A solution containing PEG diacrylate (PEGDA), NaIO4 (0.5 mM) as an
oxygen scavenger, and benzyl alcohol (0.5 wt %) as a chain transfer agent
was loaded into the channel, which was then irradiated with UV light (100
W, 365 nm, Ted Pella, Inc.).

[0170]While illustrative embodiments have been illustrated and described,
it will be appreciated that various changes can be made therein without
departing from the spirit and scope of the invention.